The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein encoded by the EGFR/ERBB1/HER1 proto-oncogene. Sequence analysis has demonstrated that the human EGFR gene is the cellular homolog of the v-ERBB1 oncogene from the avian erythroblastosis retrovirus (Downward et al., Nature, 307, 521 (1984); Ullrich et al., Nature, 309, 418 (1984)). A family of c-ERBB related cell surface receptor tyrosine kinases has been identified. The four members of the ERBB proto-oncogene family are: ErbBl/EGFR/HER1, ErbB2Neu/HER2 (Coussens et al., Science, 230, 1132 (1988)); ErbB3/HER3 (Kraus et al., Proc. Nat'l. Acad. Sci, USA, 86, 9193 (1989)); Plowman, et al., Proc. Nat'l. Acad. Sci. USA 87, 4905 (1990)); and ErbB4/HER4 (Plowman et al., Proc. Nat'l. Acad. Sci. USA, 90, 1746 (1993)). The Epidermal Growth Factor Receptor (EGFR/ErbBl/HER1) includes three functional domains: an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The extracellular domain can be further divided into four subdomains (I-IV), including two cysteine-rich regions (II and IV) and two flanking regions (I and III) (Lax et al., Cell Regul. 2,337 (1991)). Subdomains I and III are involved in ligand binding. Ligand binding to the receptor represents the first event in a complex phosphorylation cascade that culminates in DNA synthesis and cell division, as well as cell survival and metastasis.
The full-length 170 kDa human EGFR is encoded by two alternatively spliced transcripts of 5.8 and 10.5 kb (Ullrich et al., supra). In addition, alternatively spliced mRNA's from the EGFR/ErbBl/HER1 gene encode soluble forms of this receptor. A 60 kDa to 80 kDa sEGFR isoform present in human placenta extracts is encoded by an alternatively spliced 1.8 kb RNA transcript (Ilekis et al., Mol. Reprod. Devel., 41, 149 (1995); Reiter and Maihle, Nucl. Acids Res., 24, 4050 (1996)). Soluble EGF receptors also arise from aberrant transcription products in carcinoma-derived cell lines, as exemplified by the epidermoid carcinoma line, A431 (Ullrich et al., supra). In this cell line, the EGFR gene is amplified and rearranged, and a 2.8 kb transcript arises from a translocation between the 5′-region of the EGFR gene and an unidentified region of genomic DNA (Ulrich et al., supra; Merlino et al., Mol. Cell. Biol., 5, 1722 (1985); Hunts et al., Cell Mol. Genet., 11, 477 (1988)). Alternative EGFR/ErbB1 RNA transcripts of approximately 1.8-2.8 kb that encode soluble receptor isoforms comprised of the extracellular ligand binding domain and unique carboxy-terminal amino acid sequences are found in normal human, chicken, rat and mouse tissues (Maihle et al. Proc. Nat'l Acad. Sci. USA, 88, 1825 (1991); Petch et al., Mol. Cell. Biol., 10, 2973 (1990); Flickinger et al., Mol. Cell. Biol., 12,883 (1991); Das et al., Endocrinology 134,971 (1994); Rho et al., Mol. Carcinogenesis. 11, 19 (1994); Reiter and Maihle, Nucl. Acids Res., 24, 4050 (1996); Tong et al., Endocrinology 137,1492 (1996)). Conversely, proteolytically generated isoforms of sEGFR have only been observed in transfected cell lines and human carcinoma cell lines that highly over express the full-length EGF receptor (Brakebusch et al., J. Biol. Chem. 269, 32488 (1994); Zhen et al., Biochemistry 42, 5478 (2003)).
Soluble isoforms of ErbB (sErbB) receptors are being investigated in connection with several human cancers (McKenzie, Biochim. Biophys. Acta, 1072, 193 (1991); Brandt-Rauf Mutat. Res., 333, 203 (1995)). Immunoassay studies show that sErbB2 proteins are elevated in serum samples of patients with breast and ovarian cancer compared to healthy women (Mori et al., Jpn. J. Cancer. Res., 81, 489 (1990); Meden et al., Anticancer Res., 17, 757 (1997)). Recent studies suggest that low pretreatment serum sErbB2 levels are positive predictors of responsiveness to hormonal therapy for patients with metastatic breast cancer (Hayes et al., Breast Cancer Treat., 14, 135, (1993); Leitzel et al., J. Clin. Oncol., 13, 1129 (1995); Yamauchi et al., J. Clin. Oncol., 15, 2518 (1997)). Meden et al. (supra) have reported a positive association between elevated serum p105 sErbB2 levels and shorter survival for patients with stage I through IV epithelial ovarian cancer (EOC).
Immunoassay studies also show that the extracellular domain of EGFR/ErbB1 is detectable and increased in the serum of patients with asbestosis-induced lung cancer (Partanen et al., J. Occup. Med., 36, 1324 (1994); Partanen et al., Int. J. Oncol., 4, 1025 (1994)) and in the urine of patients with squamous cell carcinomas of the head, neck, and lung (Witters et al., Clin. Cancer Res., 1, 551 (1995)). In addition, U.S. Pat. No. 5,674,753, issued Oct. 7, 1997, associates increased levels of the EGFR ectodomain in blood, plasma, or serum with cancer. Ilekis et al. (Gynecol. Oncol., 65, 36 (1997)) have recently observed a positive association between expression of a p60/p80 sEGFR protein and full-length EGFR/ErbB1 in tissue samples of serous cystadenocarcinomas of the ovary. Furthermore, the EGF receptor has been shown to be over-expressed in various human tumor cell lines and neoplasms (Xu et al., Proc, Nat'l. Acad. Sci, 81, 7308 (1984); Salomon et al., Crit. Rev. Oncol. Hematol., 19, 183 (1995)), including cancers of the breast (King et al., Science, 229, 974 (1985)), lung (Hendler et al., Proc. Am. Soc. Clin. Oncol., 8, 223 (1989); Veale et al., Cancer Res., 49, 1313 (1989)), brain (Schlegel et al., Int. J. Cancer, 56, 72 (1994)), bladder (Neal et al., Cancer, 65, 1619 (1990); Mellon et al., 153, 919 (1995)), and ovary (Berchuck et al., Am. J, Obstet. Gynecol., 164, 669 (1991); Scambia et al., J. Clin. Oncol., 10, 529 (1992)).
Research concerning the biological function and clinical utility of EGF/ErbB receptors has provided contradictory results, and has not yet provided a clear indication of how any particular ErbB receptor can be used as a tool for the risk assessment and prevention, screening and early detection, diagnosis, prognosis, theragnosis, and treatment of any particular cancer type. Therefore, a need exists for the isolation and characterization of soluble epidermal growth factor receptor protein molecules and their isoforms in human tissues, and for the analysis/quantitation of these proteins in normal versus pathological conditions. In this regard, a useful, quantitative method to detect the presence of biologically relevant, specific EGF receptor isoform expression in human body fluids and tissues, and for discerning the onset and progression of diseases associated with these soluble epidermal growth factor receptor protein molecules is needed.